Abstract
Purpose :
The purpose of these experiments was to investigate how NR2F1 can influence retinal development through transcriptomic and genomic manipulation. These data expand upon the previous work to characterize NR2F1’s cell fate specification phenotype. It also sheds light on NR2F1’s gene regulatory network and its role within it. The goal is to learn how NR2F1 influences how certain cell types, such as cone photoreceptors, are originally generated. This could provide insight into regenerating these cell types if lost in disease pathology.
Methods :
Human NR2F1 was cloned into the pCAGIG overexpression construct. Alongside a GFP only control construct, P0 CD1 wild type murine retinas were electroporated and cultured. For transcriptomic experiments, retinas were cultured for two or five days. For scATAC-seq profiling, retinas were cultured for two days. GFP+ cells were isolated utilizing fluorescence activated cell sorting and then processed using 10x genomics scRNA and scATAC kits. The ATAC samples were multiplexed using ScaleBio’s kit. Seurat and Signac were utilized to analyze the sequencing data produced.
Results :
NR2F1 was able to influence late stage retinal progenitor cell fate specification. This was evident in the transcriptomic changes seen in the NR2F1 electroporated cells compared to GFP+ controls. Even more interesting was the evidence that cone photoreceptors were being born from late stage RPCs outside their temporal window shown through pseudotime analysis. The genomic organization of retinal cell types was influenced by the ectopic overexpression of NR2F1. Retinal progenitor cells and neurogenic progenitors showed changes in genomic loci access compared to the control sample.
Conclusions :
NR2F1 is capable of influencing the cell fate specification of late stage retinal progenitor cells through ectopic overexpression. Cells are diverted away from late stage cell fates and towards early born cell fates suggesting large reprogramming capacity. This was reflected in the transcriptomic and genomic organization changes in the NR2F1 treated samples in the scRNA and scATAC-seq data.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.